Background and Purpose Thrombo-inflammation is a key feature of stroke pathophysiology and provides multiple candidate drug targets. Thrombin exerts coagulation-independent actions via protease-activated receptors (PAR), of which PAR1 has been implicated in stroke-associated neuro-inflammation. The role of PAR4 in this context is less clear. This study examined if the selective PAR4 antagonist ML354 provides neuroprotection in experimental stroke and explored the underlying mechanisms. Experimental Approach Mouse primary cortical neurons were exposed to oxygen-glucose deprivation (OGD) and simulated reperfusion ± ML354. For comparison, functional Ca2+-imaging was performed upon acute stimulation with a PAR4 activating peptide or glutamate. Male mice underwent sham operation or transient middle cerebral artery occlusion (tMCAO), with ML354 or vehicle treatment beginning at recanalization. A subset of mice received a platelet-depleting antibody. Stroke size and functional outcome were assessed. Abundance of target genes, proteins and cell markers was determined in cultured cells and tissues by qPCR, immunoblotting and immunofluorescence. Key Results Stroke upregulates PAR4 expression in cortical neurons in vitro and in vivo. OGD augments spontaneous and PAR4-mediated neuronal activity; ML354 suppresses OGD-induced neuronal excitotoxicity and apoptosis. ML354 applied in vivo after tMCAO reduces infarct size, apoptotic markers, macrophage accumulation and interleukin-1β expression. Platelet depletion did not affect infarct size in mice with tMCAO ± ML354. Conclusions and Implications Selective PAR4 inhibition during reperfusion improves infarct size and neurological function after experimental stroke by blunting neuronal excitability, apoptosis and local inflammation. PAR4 antagonists may provide additional neuroprotective benefits in patients with acute stroke beyond their canonical antiplatelet action.

Commentary on: Activation of Nrf2 signalling pathway by tectoridin protects against ferroptosis in particulate matter-induced lung injuryWenfu Cao1, Xinrui Guo1, Xinyu Li, Dapeng Chen1*1Compartive medicine, Dalian Medical University, Dalian city, 116044, Liaoning province, China.*Corresponding author: Dapeng Chen, Comparative Medicine Department of Researching and Teaching, Dalian Medical University, Dalian 116044, P. R. China, Tel/Fax: +86 411 86110862; Email: [email protected]: lung injury, cell death, PM2.5, drug discovery, ferroptosisWe read with great interest the article entitled ”Activation of Nrf2 signalling pathway by tectoridin protects against ferroptosis in particulate matter-induced lung injury ” by Dong et al. in the British Journal of Pharmacology (Donget al. , 2023).The authors demonstrate that tectoridin has a therapeutic effect on particulate matter-induced lung injury (PM2.5-induced lung injury), and they believe that, this treatment mechanism is related to the inhibition of ferroptosis through targeting Nrf2. There are varying opinions regarding the future of targeted ferroptosis inhibition in the treatment of PM2.5-induced lung injury, and its clarity remains uncertain.With the rapid economic development, air pollution has emerged as the fifth most significant risk factor for all-cause mortality on a global scale, imposing a substantial economic burden. The animal model of lung injury, commonly employed in fundamental studies, involves the inhalation of PM2.5. In the case of lung injury induced by PM2.5, oxidative stress and inflammation, which are well-recognized indicators of lung injury, closely mirror the clinical symptoms of such injury. To induce severe and easily measurable markers of lung injury, mice were exposed to highly concentrated levels of PM2.5. Prolonged exposure to PM2.5 instigates the overproduction of reactive oxygen species (ROS), precipitating oxidative stress that results in apoptosis, and in more severe cases, necrosis (Zhao et al. , 2022).The signaling pathways involved in PM2.5-induced lung injury encompass cell death (apoptosis, necrosis and pyroptosis), inflammation, and increased oxidative stress(He et al. , 2021). Extensive research has factually demonstrated that compounds exhibiting anti-inflammatory and antioxidant characteristics are beneficial in alleviating PM2.5-induced lung injury. It is well known that Nrf2 activation can provide relief for lung injury through anti-inflammatory, antioxidant, and anti-apoptotic effects. Based on the current findings presented in the article by Dong et al , it is challenging to prove that tectoridin-induced treatment is directly associated with ferroptosis inhibition in PM2.5-induced lung injury. In my opinion, the conclusion should state that tectoridin administration alleviated ferroptosis in PM2.5-induced lung injury, as it remains unclear whether ferroptosis is the cause or result of PM2.5-induced lung injury. I apologize if my previous wording was not entirely accurate; it was not intended as a mere play on words, but rather an attempt at precision.Targeted inhibition of cell death, without affecting inflammation and oxidative stress, has never proven to be a satisfactory option for alleviating tissue injury. In PM2.5-induced lung injury, apoptosis is the primary form of cell death and is typically a direct consequence of excessive oxidative stress following PM2.5 exposure . However, the direct inhibition of apoptosis using caspase inhibitors has some palliative effects and is not an ideal treatment (Liu et al. , 2016).Ferroptosis, on the other hand, is a form of regulated cell death triggered by lipid peroxidation. Unlike apoptosis, it does not follow the typical apoptotic pathway and possesses immunogenic properties. The classic biochemical marker of ferroptosis is GPX4, SLC7A11, ATP5G3, and NOCA4. Metabolic disruptions in lipids, iron, and amino acids are closely associated with the activation of ferroptosis. Therefore, in this discussion, we will carefully explore the effectiveness of targeted ferroptosis inhibition in alleviating PM2.5-induced lung injury.Firstly, there is still a lack of large-scale clinical research to provide evidence for the causal relationship between ferroptosis and PM2.5-induced lung injury. Many current conclusions are obtained from animal or cell studies. While apoptosis and necrosis are the predominant cell death types in PM2.5-induced lung injury, recent studies have also reported other types of cell death, including necroptosis and ferroptosis. The association between ferroptosis and PM2.5-induced lung injury has only recently been observed, and further investigation is needed to establish the definitive causality. There are suggestions that PM2.5 may play a role in inducing death of lung epithelial cells through its influence on the balance of iron overload, lipid peroxidation, and redox reactions. However, whether ferroptosis is the main cause and promise target of PM2.5 induced lung injury requires further study. A search conducted on PubMed using the keywords ”PM2.5; acute lung injury; ferroptosis” yielded approximately 13 publications between 2021 and 2024. Unfortunately, none of these studies provide conclusive evidence supporting ferroptosis as the primary pathological mechanism or promise target of PM2.5-induced lung injury. The lack of precise and convincing experimental designs and results undermines the conclusion that inhibiting ferroptosis contributes to the alleviation of PM2.5-induced lung injury.Secondly, the utilization of solely biochemical markers of ferroptosis to ascertain the presence or participation of ferroptosis is subject to debate, as autophagy, apoptosis, and oxidative stress exhibit overlapping characteristics with ferroptosis, potentially leading to a pseudo-manifestation of ferroptosis caused by other factors. Given these considerations, conducting a comprehensive study on the essence of ferroptosis in PM2.5 induced lung injury without the involvement of additional factors becomes a challenging endeavor.Lastly, caution must be exercised when utilizing ferroptosis inhibitors due to the potential interference with the pharmacological properties of other drugs, thereby compromising the treatment objective. Studies have shown that combining anti-cancer drugs with a ferroptosis inducer enhances its anticancer efficacy. This discovery introduces a promising approach involving the combination of anti-cancer drugs with a ferroptosis inducer for tumor treatment. However, further comprehensive investigation is necessary to evaluate the application of ferroptosis inhibitors in the treatment of tumor patients with tissue injury syndrome, such as cisplatin-induced kidney injury (CP-AKI).Hu et al also reported the treatment effects of leonurine on cisplatin-induced acute kidney injury by inhibiting ferroptosis in BJP journal (Hu et al., 2022). It is likely that ferroptosis is not only a concomitant phenomenon but also a secondary event triggered by cisplatin exposure. Upon acute kidney occurrence, neutrophils are the first type of accumulated white blood cells in the kidneys. In comparison to apoptotic cells, ferroptotic cells exhibit higher immunogenicity and are capable of recruiting macrophages, which subsequently induce neutrophil recruitment. However, the results have shown that in CP-AKI, both parenchymal cells and immune cells, particularly macrophages and neutrophils, display significantly upregulated activity in the ferroptosis pathway(Chen et al., 2023). This non-specific activation poses challenges to develop targeted therapeutic strategies to alleviate CP-AKI through inhibiting ferroptosis.In conclusion, the potential for the development of ferroptosis inducers in cancer therapy holds promise. However, the effectiveness of ferroptosis inhibitors in treating PM2.5 induced lung injury or other forms of acute tissue injury remains uncertain. This correspondence should not be interpreted as a negative critique of the article by Dong et al., who have astutely observed the mitigating effects of tectoridin on ferroptosis in PM2.5-induced lung injury and have provided a probable connection between Nrf2 activation and ferroptosis inhibition. Conversely, our aim in writing this correspondence is to draw greater attention from researchers towards exploring the causal relationship between ferroptosis and acute tissue injury.AUTHOR CONTRIBUTIONSDapeng Chen: Conceptualization; Wenfu CAO, Xinrui Guo, and Xinyu Li: writing original draft; Dapeng Chen and Wenfu Cao: writing, review, and editing.CONFLICT OF INTEREST STATEMENTThe authors have no conflicts of interest to declare.ORCIDDapeng Chen: https://orcid.org/0000-0001-9838-1646REFERENCESChen Z, Li Y, Yuan Y, Lai K, Ye K, Lin Y, et al. (2023). Single-cell sequencing reveals homogeneity and heterogeneity of the cytopathological mechanisms in different etiology-induced AKI. Cell Death Dis 14 (5):318.Dong T, Fan X, Zheng N, Yan K, Hou T, Peng L, et al. (2023). Activation of Nrf2 signalling pathway by tectoridin protects against ferroptosis in particulate matter-induced lung injury. Br J Pharmacol 180 (19): 2532-2549.Hu J, Gu W, Ma N, Fan X, Ci X (2022). Leonurine alleviates ferroptosis in cisplatin-induced acute kidney injury by activating the Nrf2 signalling pathway. Br J Pharmacol179 (15): 3991-4009.Liu M, Shi L, Zou X, Zheng X, Zhang F, Ding X, et al. (2016). Caspase inhibitor zVAD-fmk protects against acute pancreatitis-associated lung injury via inhibiting inflammation and apoptosis. Pancreatology16 (5): 733-738.Zhao C, Pu W, Wazir J, Jin X, Wei L, Song S, et al. (2022). Long-term exposure to PM2.5 aggravates pulmonary fibrosis and acute lung injury by disrupting Nrf2-mediated antioxidant function. Environ Pollut 313: 120017.

Vascular smooth muscle cell (vSMC) dysfunction is a critical contributor to cardiovascular diseases, including atherosclerosis, restenosis, and vein graft failure. Recent advances have unveiled a fascinating breadth of non-coding RNAs (ncRNAs) that play a pivotal role in regulating vSMC function. This review aims to provide an in-depth analysis of the mechanisms underlying vSMC dysfunction and the therapeutic potential of various ncRNAs in mitigating this dysfunction, either preventing or reversing it. We explore the intricate interplay of microRNAs, long-non-coding RNAs and circular RNAs, shedding light on their roles in regulating key signalling pathways associated with vSMC dysfunction. Moreover, we discuss the prospects and challenges associated with developing ncRNA based therapies for this prevalent cardiovascular pathology.

Editorial. Platelet purinergic receptors and non-thrombotic diseases.Simon C. Pitchford1* and Dingxin Pan.11Pulmonary Pharmacology Unit, Institute of Pharmaceutical Science, King’s College London, London, UK.*Author for correspondence and reprint requests:Dr Simon PitchfordPulmonary Pharmacology UnitInstitute of Pharmaceutical Science5.43 Franklin Wilkins Building150 Stamford StreetWaterloo CampusKing’s College LondonLondon UKSE1 9NHPhone: +44 2078484266Fax: +44 207 [email protected]

Editorial on the themed issue:

Background and Purpose: Our previous study reported that erythroferrone (ERFE), a newly identified hormone produced by erythroblasts, responded to recombinant human erythropoietin (rHuEPO) sensitively but its dynamics was complicated by double peaks and circadian rhythm. This study intends to elucidate the underlying mechanisms for the double peaks of ERFE dynamics, and further determine whether early ERFE measurements can predict hemoglobin (HGB) responses to rHuEPO. Experimental Approach: By expressing recombinant rat ERFE protein and investigating its deposition in rats, the production of ERFE was deconvoluted. To explore the role of iron in ERFE production, we monitored short-term changes of iron status after injection of rHuEPO or deferiprone (DFP). Pharmacokinetic/pharmacodynamic (PK/PD) modelling was used to confirm the mechanisms and examine the predictive ability of ERFE for long-term HGB responses. Key Results: The rRatERFE protein was successfully expressed and purified. The production of ERFE was deconvoluted and showed two independent peaks (2 h and 8 h). Transient iron decrease was observed at 4h after rHuEPO injection and DFP induced significant increases of ERFE. Based on this mechanism, the PK/PD model could characterize the complex dynamics of ERFE. In addition, the model predictions further revealed a stronger correlation between ERFE and HGB peak values than that for observed values. Conclusions and Implications: The complex dynamics of ERFE should be composited by an immediate release and transient iron deficiency-mediated secondary production of ERFE. The early peak values of ERFE, which occur within a few hours, can predict HGB responses several weeks after ESA treatment.

Biased signaling is a natural result of GPCR allosteric function and should be expected from any and all synthetic agonists. Therefore, it may be encountered in all agonist discovery projects and must be considered as a beneficial (or possible detrimental) feature of new candidate molecules. While bias is easily detected , the synoptic nature of GPCR signaling makes translation of simple in vitro bias to complex in vivo systems problematic. The practical outcome of this is a difficulty in predicting the therapeutic value of biased signaling due to the failure of translation of identified biased signaling to in vivo agonism. This is discussed in this review as well as some new ways forward to improve this translation process and better exploit this powerful pharmacologic activity.

Background and Purpose: P2X4 receptors (P2X4R) are ligand gated cation channels that are activated by extracellular adenosine 5′-triphosphate (ATP) released by neurons and glia. The receptors are widely expressed in the brain and have fractional calcium currents comparable to NMDA receptors. Although P2X4Rs were described to modulate synaptic transmission and plasticity, their involvement in shaping neuronal network activity remains to be elucidated. Exp. Approach: We investigated the effects of P2X receptors on network and synaptic level using local field potential electrophysiology, whole cell patch clamp recordings and calcium imaging in fast spiking parvalbumin positive interneurons (PVINs) in rat and mice hippocampal slices. The stable ATP analogue ATPγS, selective antagonists and P2X4R knockout mice were used. Key results: The P2XR agonist ATPγS reversibly decreased the power of gamma oscillations. This inhibition could be antagonized by the selective P2X4R antagonist PSB-12062 and was not observed in P2X4-/- mice. The phasic excitatory inputs of CA3 PVINs were one of the main regulators of the gamma power. Associational fibre compound excitatory postsynaptic currents (cEPSCs) in CA3 PVINs were inhibited by P2X4R activation. This effect was reversible, dependent on intracellular calcium and dynamin-dependent internalization of AMPA receptors. Conclusions and Implications: The results indicate that P2X4Rs are an important source of dendritic calcium in CA3 PVINs, thereby regulating excitatory synaptic inputs onto the cells and the state of gamma oscillations in the hippocampus. P2X4Rs represent an effective target to modulate hippocampal network activity in pathophysiological conditions such as Alzheimer’s disease and schizophrenia.

G protein-coupled receptors (GPCRs) are a large family of cell surface receptors that play a critical role in nervous system function by transmitting signals between cells and their environment. They are involved in many, if not all, nervous system processes, and their dysfunction has been linked to various neurological disorders representing important drug targets. In this review, we will first discuss the role of the nervous system GPCRs in the modulation of tripartite synapse function and how GPCRs control energy metabolism in the brain. We will then discuss the (patho)physiology and pharmacology of opioid, cannabinoid, acetylcholine, chemokine, and melatonin GPCRs in the nervous system. Furthermore, we will briefly report on adhesion GPCR function in nervous tissues. Finally, we will address orphan GPCRs, their implication in the nervous system function and disease, and the challenges that need to be addressed in the future to deorphanize them.

Chronic Obstructive Pulmonary Disease (COPD) is a major incurable health burden, ranking as the 3rd leading cause of death worldwide, mainly driven by cigarette smoking. COPD is characterised by persistent airway inflammation, lung function decline, and premature aging with the presence of pulmonary senescent cells. This review proposes that cellular senescence, a state of stable cell cycle arrest linked to ageing; induced by inflammation and oxidative stress in COPD, extends beyond the lungs and impacts the systemic circulation. This “spill over” of senescent cells contributes to brain inflammation and damage, increasing the risk of neurological comorbidities, such as stroke, cerebral small vessel disease, and Alzheimer’s disease. The review explores the role of cellular senescence in COPD-associated brain conditions and investigates the relationship between cellular senescence and circadian rhythm in COPD. Additionally, it discusses potential therapies, including senomorphic and senolytic treatments, as novel strategies to halt or improve COPD progression.

The KEAP1-NRF2 system plays a central role in cytoprotection and defense mechanisms against oxidative stress. Because KEAP1 serves as a biosensor for electrophiles by using its reactive thiols and because NRF2 is a transcriptional factor regulating genes involved in the sulfur-mediated redox reactions, the KEAP1-NRF2 system has been regarded as a sulfur-utilizing cytoprotective mechanism. NRF2 is a key regulator of cytoprotective genes, such as antioxidant and detoxification genes, and also to possess potent anti-inflammatory activity. NRF2 has been recently focused as a great modifier/regulator for the cellular metabolism and mitochondrial function. Particularly, the NRF2-mediated regulatory mechanisms of metabolites and mitochondria has been considered diverse, but has not been fully-clarified yet. This review article provides an overview of the molecular mechanisms that regulate NRF2 signaling and its cytoprotective roles, and also highlights NRF2 contribution to the cellular metabolism, particularly in the context of mitochondrial function and newly found sulfur metabolism.

Background and purpose Chymotrypsin is a serine protease produced by the pancreas and secreted into the lumen of the small intestine, where it digests food proteins. Due to its presence in the gut lumen, we hypothesized that chymotrypsin activity may be found close to epithelial cells and signals to them via Protease-activated receptors (PARs). We deciphered molecular pharmacology mechanisms for chymotrypsin signaling in intestinal epithelial cells. Experimental approaches The presence and activity of chymotrypsin were evaluated by western blot (WB) and enzymatic activity tests in luminal and mucosal compartments of murine and human gut samples. The ability of chymotrypsin to cleave the extracellular domain of PAR1 or PAR2 was assessed using cell lines expressing N-terminally-tagged receptors. The cleavage site of chymotrypsin on PAR1 and PAR2 was determined by HPLC-MS analysis. To study the pharmacology of chymotrypsin signals, we investigated calcium signaling and ERK1/2 activation using calcium mobilization assays and WB in CMT93 intestinal epithelial cells. Key results We found that chymotrypsin was present and active in the vicinity of the murine and human colonic epithelium. Molecular pharmacology studies evidenced that chymotrypsin cleaved both PARs receptors. While chymotrypsin activated calcium and ERK1/2 signaling pathways through PAR2, it disarmed PAR1, preventing further activation by its canonical agonist thrombin. CONCLUSION Our work suggests that the function of chymotrypsin in the gut lumen goes well beyond a simple digestive role. Our results highlight the ability of chymotrypsin to signal to intestinal epithelial cells via PARs, which may have important physiological consequences in gut homeostasis.

Background and Purposes: Chemotherapy-induced peripheral neuropathy commonly causes neuropathic pain. The pathogenesis of CIPN is unclear, and effective therapies are also lacking. Naringenin, a dihydroflavonoid compound in Rutaceae plants and citrus fruits, has anti-inflammatory, antioxidant, and anti-tumor activities. However, its effect on chemotherapy-induced pain has not been investigated. Experimental Approach: We used Paclitaxel (PTX) to establish a mouse model of chemotherapy-induced pain. Mechanical and thermal pain thresholds, glial activation, calcitonin gene-related peptide (CGRP) expression, c-fos expression, phosphorylation of nuclear factor κB (NF-κB), dorsal root ganglion (DRG) neuron excitability, and cell survival of pancreatic, colorectal, and gastric cancer cell lines were measured. Key Results: Systemic application of Naringenin reduced the mechanical and thermal pain hypersensitivity induced by PTX. Naringenin reduced the activation of glial cells in both DRGs and the spinal dorsal horn of PTX-treated mice. Naringenin decreased the PTX-enhanced CGRP expression in DRG and spinal neurons. Naringenin reversed the PTX-enhanced c-fos expression and excitability of DRG neurons. Naringenin downregulated PTX-elevated NF-κB phosphorylation in the spinal cord. Additionally, co-administration of Naringenin with PTX enhanced the inhibitory effect of PTX on pancreatic and colorectal cancer cell growth, whereas the application of Naringenin alone inhibited the survival of pancreatic cancer cells. Conclusion and Implications: Naringenin alleviates PTX-induced pain and may facilitate PTX’s anti-tumor effect. The mechanism involves the inhibition of glial activation, CGRP production, and neuronal sensitization in PTX-treated mice. Our study suggests the multiple beneficial actions of Naringenin in chemotherapy by mitigating side effects and inhibiting tumor growth.

Background & purpose Morphine is important for treatment of acute and chronic pain. However, there is high interpatient variability and often inadequate pain relief and adverse effects. To better understand variability in the dose effect relationships of morphine, we investigated the impact of its nonlinear blood-brain-barrier (BBB) transport on mu-opioid receptor (μ-OR) occupancy in different CNS locations, in conjunction with its main metabolites that bind to the same receptor. Methods CNS exposure profiles for morphine, M3G and M6G for clinically relevant dosing regimens based on intravenous, oral immediate- and extended-release formulations were generated using a CNS PBPK model which incorporated nonlinear BBB transport of morphine. The simulated CNS exposure profiles were then used to derive corresponding μ-OR occupancies at multiple CNS pain matrix locations. Results The simulated CNS exposure profiles for morphine, M3G and M6G, associated with nonlinear BBB transport of morphine resulted in varying μ-OR occupancies between the different dose regimens, formulations, and CNS locations. We find that at lower doses, the μ-OR occupancy of morphine was relatively higher than at higher doses of morphine, due to the relative contribution of M3G and M6G. At such higher doses, M6G showed higher occupancy than morphine, whereas M3G occupancy was low throughout the dose ranges. Conclusion and implications Nonlinear BBB transport of morphine influences the μ-OR occupancy ratios of morphine and its metabolites, depending on dose and route of administration, and CNS location. This may impact the clinical effects of morphine treatment for pain relief.

Background and Purpose: 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) catalyzes the oxoreduction of cortisone to cortisol, thereby amplifying levels of active glucocorticoids. It is considered a pharmaceutical target in metabolic disease and cognitive impairments. 11β-HSD1 also converts some 7oxo-steroids to their 7β-hydroxy forms. A recent study in mice described the ratio of tauroursodeoxycholic acid (TUDCA)/tauro-7oxolithocholic acid (T7oxoLCA) as a biomarker for decreased 11β-HSD1 oxoreductase activity. The present study aimed to evaluate the equivalent bile acid ratio glycoursodeoxycholic acid (GUDCA)/glyco-7oxolithocholic acid (G7oxoLCA) as a biomarker for pharmacological 11β-HSD1 inhibition in humans and compare it with the currently applied urinary (5α-tetrahydrocortisol+tetrahydrocortisol)/tetrahydrocortisone ((5αTHF+THF)/THE) ratio. Experimental Approach: Bile acid profiles were analyzed by ultra-HPLC tandem-MS in blood samples from two independent, double-blind placebo-controlled clinical studies on the orally administered selective 11β-HSD1 inhibitor AZD4017. The blood GUDCA/G7oxoLCA ratio was compared with the urinary tetrahydro-glucocorticoid ratio for the ability to detect 11β-HSD1 inhibition. Key Results: No significant alterations were observed in the bile acid profiles following 11β-HSD1 inhibition by AZD4017, except for an increase of the secondary bile acid G7oxoLCA. The enzyme product/substrate ratio GUDCA/G7oxoLCA was found to be more reliable to detect 11β-HSD1 inhibition than the absolute G7oxoLCA concentration in both cohorts. Comparison of the blood GUDCA/G7oxoLCA ratio with the urinary (5αTHF+THF)/THE ratio revealed that both ratios successfully detect 11β-HSD1 inhibition. Conclusion and Implications: 11β-HSD1 inhibition does not cause major alterations in bile acid homeostasis. The GUDCA/G7oxoLCA ratio represents the first blood biomarker of pharmacological 11β-HSD1 inhibition and may replace or complement the urinary (5αTHF+THF)/THE ratio biomarker.

Background and Purpose: Peripheral nerve trauma-induced dysregulation of pain-associated genes in the primary sensory neurons of dorsal root ganglion (DRG) contributes to neuropathic pain genesis. RNA-binding proteins participate in gene transcription. We hypothesized that RALY, an RNA-binding protein, participated in nerve trauma-induced dysregulation of DRG pain-associated genes and nociceptive hypersensitivity. Methods and results: Immunohistochemistry staining showed that RALY was expressed exclusively in the nuclei of DRG neurons. Peripheral nerve trauma caused by chronic constriction injury (CCI) of unilateral sciatic nerve produced time-dependent increases in the levels of Raly mRNA and RALY protein in injured DRG. Blocking this increase through DRG microinjection of adeno-associated virus 5 (AAV5)-expressing Raly shRNA reduced the CCI-induced elevation in the amount of eukaryotic initiation factor 4 gamma 2 (eIF4G2) mRNA and eIF4G2 protein in injured DRG and mitigated the development and maintenance of CCI-induced nociceptive hypersensitivity, without altering basal (acute) response to noxious stimuli and locomotor activity. Mimicking DRG increased RALY through DRG microinjection of AAV5 expressing Raly mRNA upregulated the expression of eIF4G2 mRNA and eIF4G2 protein in the DRG and led to hypersensitive responses to noxious stimuli in the absence of nerve trauma. Mechanistically, CCI promoted the binding of RALY to the promoter of eIF4G2 gene and triggered its transcriptional activity. Conclusion and Implications: Our findings indicate that RALY participates in nerve trauma-induced nociceptive hypersensitivity likely through transcriptionally triggering eIF4G2 expression in the DRG. RALY may be a potential target in neuropathic pain management.

Background and Purpose Development of core concepts in disciplines such as biochemistry, microbiology, and physiology transformed teaching. They provided the foundation for the development of teaching resources for global educators, as well as valid and reliable approaches to assessment. An international research consensus recently identified 25 core concepts of pharmacology. The current study aimed to define and unpack these concepts. Experimental approach A two-phase, iterative approach, involving 60 international pharmacology education experts was used. The first phase involved drafting definitions for the core concepts and identifying key sub-concepts via a series of online meetings and asynchronous work. These were refined in the second phase, through a two-day hybrid workshop followed by a further series of online meetings and asynchronous work. Key Results The project produced consensus definitions for a final list of 24 core concepts and 103 sub-concepts of pharmacology. The iterative, discursive methodology resulted in the modification concepts from the original study, including the change of ‘drug-receptor interaction’ to ‘drug-target interaction’ and the change of the core concept ‘agonists and antagonists’ to sub-concepts of drug-target interaction. Conclusion and Implications The definitions and sub-concepts of the 24 core concepts provide an evidence-based foundation for pharmacology curricula development and evaluation. The next steps for this project include the development of a concept inventory to assess acquisition of the concepts, as well as the development of cases studies and educational resources to support teaching by the global pharmacology community, and student learning of the most critical and fundamental concepts of the discipline.

Background and Purpose: Sepsis-surviving adult individuals commonly develop immunosuppression and increased susceptibility to secondary infections, outcome mediated by the axis IL-33/ILC2s/M2 macrophages/Tregs. Nonetheless, the long-term immune consequences of pediatric sepsis are indeterminate. We sought to investigate the role of age in the genesis of immunosuppression following sepsis. Experimental Approach: Here, we compared the frequency of Tregs, the activation of the IL33/ILC2s axis in M2 macrophages, and the DNA methylation of epithelial lung cells from post-septic infant and adult mice. Likewise, sepsis-surviving mice were inoculated intranasally with Pseudomonas aeruginosa or by subcutaneous inoculation of the B16 melanoma cell line. Finally, blood samples from sepsis-surviving patients were collected and the concentrations of IL-33 and Tregs frequency were assessed. Key Results: In contrast to 6-week-old, 2-week-old mice were resistant to secondary infection and did not show impairment in tumour controls upon melanoma challenge. Mechanistically, increased IL-33 levels, Tregs expansion, and activation of ILC2s and M2-macrophages were observed in 6-week-old but not 2-week-old post-septic mice. Moreover, impaired IL-33 production in 2-week-old post-septic mice was associated with increased DNA methylation in lung epithelial cells. Notably, IL-33 treatment boosted the expansion of Tregs and induced immunosuppression in 2-week-old mice. Clinically, adults but not pediatric post-septic patients exhibited higher counts of Tregs and sera IL-33 levels. Conclusion and Implications: These findings demonstrate a crucial and age-dependent role for IL-33 in post-sepsis immunosuppression. Thus, a better understanding of this process could lead to differential treatments for adult and pediatric sepsis.